Department of Chemistry, The University of Western Ontario, London, Ontario N6A 5B7, Canada.
J Phys Chem B. 2021 Apr 22;125(15):3803-3814. doi: 10.1021/acs.jpcb.1c00944. Epub 2021 Apr 13.
Electrosprayed protein ions can retain native-like conformations. The intramolecular contacts that stabilize these compact gas-phase structures remain poorly understood. Recent work has uncovered abundant salt bridges in electrosprayed proteins. Salt bridges are zwitterionic BH/A contacts. The low dielectric constant in the vacuum strengthens electrostatic interactions, suggesting that salt bridges could be a key contributor to the retention of compact protein structures. A problem with this assertion is that H are mobile, such that H transfer can convert salt bridges into neutral B/HA contacts. This possible salt bridge annihilation puts into question the role of zwitterionic motifs in the gas phase, and it calls for a detailed analysis of BH/A versus B/HA interactions. Here, we investigate this issue using molecular dynamics (MD) simulations and electrospray experiments. MD data for short model peptides revealed that salt bridges with static H have dissociation energies around 700 kJ mol. The corresponding B/HA contacts are 1 order of magnitude weaker. When considering the effects of mobile H, BH/A bond energies were found to be between these two extremes, confirming that H migration can significantly weaken salt bridges. Next, we examined the protein ubiquitin under collision-induced unfolding (CIU) conditions. CIU simulations were conducted using three different MD models: (i) Positive-only runs with static H did not allow for salt bridge formation and produced highly expanded CIU structures. (ii) Zwitterionic runs with static H resulted in abundant salt bridges, culminating in much more compact CIU structures. (iii) Mobile H simulations allowed for the dynamic formation/annihilation of salt bridges, generating CIU structures intermediate between scenarios (i) and (ii). Our results uncover that mobile H limit the stabilizing effects of salt bridges in the gas phase. Failure to consider the effects of mobile H in MD simulations will result in unrealistic outcomes under CIU conditions.
电喷雾的蛋白质离子可以保留类似天然的构象。这些紧凑气相结构稳定的分子内接触仍知之甚少。最近的工作揭示了电喷雾蛋白质中丰富的盐桥。盐桥是两性离子 BH/A 接触。真空中的低介电常数增强了静电相互作用，表明盐桥可能是保持蛋白质结构紧凑的关键因素。但这一说法存在一个问题，即 H 是可移动的，H 的转移可以将盐桥转化为中性的 B/HA 接触。这种可能的盐桥湮灭使两性离子基序在气相中的作用受到质疑，需要对 BH/A 与 B/HA 相互作用进行详细分析。在这里，我们使用分子动力学 (MD) 模拟和电喷雾实验来研究这个问题。短模型肽的 MD 数据表明，具有静态 H 的盐桥的离解能约为 700kJ/mol。相应的 B/HA 接触弱 1 个数量级。当考虑到移动 H 的影响时，发现 BH/A 键能介于这两个极端之间，这证实了 H 的迁移可以显著削弱盐桥。接下来，我们在碰撞诱导解折叠 (CIU) 条件下研究了蛋白质泛素。使用三种不同的 MD 模型进行了 CIU 模拟：（i）具有静态 H 的阳性运行不允许形成盐桥，并产生高度扩展的 CIU 结构。（ii）具有静态 H 的两性离子运行导致大量盐桥形成，最终导致更紧凑的 CIU 结构。（iii）移动 H 的模拟允许盐桥的动态形成/湮灭，产生介于（i）和（ii）方案之间的 CIU 结构。我们的结果表明，移动 H 限制了盐桥在气相中的稳定作用。在 MD 模拟中不考虑移动 H 的影响，将导致在 CIU 条件下产生不现实的结果。
